Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Summary

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Abstract

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and fertilization. However, identifying suitable varieties to serve as pollinizers is a time-consuming process. To solve this problem, we have developed a rapid method for identifying pollination-compatible citrus cultivars that utilizes agarose gel electrophoresis and aniline blue staining. Pollen compatibility is determined based on the identification of S genotypes by extracting total DNA and performing PCR-based genotyping assays with specific primers. Additionally, styles are collected 3-4 days after manual pollination, and aniline blue staining is performed. Finally, the growth status of the pollen tubes is observed with a fluorescence microscope. Pollen compatibility and incompatibility can be established by observing whether the pollen tube growth is normal or suppressed, respectively. Due to its simplicity and cost-effectiveness, this method is an effective tool for determining the pollen compatibility and incompatibility of different citrus varieties to establish incompatibility groups and incompatibility relationships between different cultivars. This method provides information essential for the successful selection of suitable pollinizer trees and, thus, facilitates the establishment of new orchards and the selection of appropriate parents for breeding programs.

Introduction

Self-incompatibility (SI) is a genetically controlled mechanism present in approximately 40% of angiosperm species. In this process, the pistil rejects pollen from a plant with the same SI genotype and, thus, prevents self-fertilization^1,2. Ma jia pummelo is a local variety in Jinagsu province, China, with the excellent qualities of large, pink fruit, a rich juice content, a sweet and sour taste, and a thick peel³. Although SI promotes outcrossing, it negatively impacts the yield and quality of fruits⁴ and necessitates suitable pollinizer trees with distinct SI g....

Protocol

1. Preparation for aniline blue staining

1. Prepare the following reagents and tools for the experiment: a pollinator brush, tweezers, a pencil, sulfate paper, a pollination bag, zip lock bags, paper clips, formaldehyde, glacial acetic acid, absolute ethanol, centrifuge tubes, forceps, glue droppers, glass slides, coverslips, scalpels, and polyethylene glycol.
2. Prepare in vitro germination medium containing 0.02% MgSO₄, 0.01% KNO₃, 0.03% Ca(NO₃)₂, 0.01% H₃BO₃, 20% PEG-4000, and 15% sucrose. Adjust the pH to 6.0-6.2 with KOH. Use a magnetic stirrer as PEG-4,000....

Results

For the experiments done here, mature flowers were selected, the anthers were collected, dried in an oven, and the pollen was germinated at 28°C for 12 h. The pollen viability and germination rates were quantified as shown in Figure 1.

Citrus was manually pollinated, and the pollen compatibility and incompatibility were assessed using aniline blue staining and fluorescence microscopy. The compatible pollen could germinate on the surface of the stigma and.......

Discussion

In fruit crops, both parthenocarpy and SI are important traits because they pave the way for seedless fruits - a trait that is highly appreciated by consumers. Self-incompatibility promotes the rejection of self-pollen and, thus, prevents inbreeding²⁰. Among citrus, pummelo is a self-incompatible variety⁷. Almost 40% of all angiosperm species exhibit SI²¹. This trait prevents fruit setting, lowers the yield, and brings huge economic losses to growers.......

Materials

Name	Company	Catalog Number	Comments
absolute ethanol	Sinopharm Chemical ReagentCo., Ltd	10009218
Aniline blue	Sinopharm Chemical Reagent Co.,Ltd
Boric acid, H3BO3	Sinopharm Chemical ReagentCo., Ltd	10004818
Brown bottle	Labgic Technology Co., Ltd
Calcium nitrate tetrahydrate, Ca(NO3 )2	Sinopharm Chemical ReagentCo., Ltd	80029062
Centrifugal tube	Labgic Technology Co., Ltd
centrifuge tubes	Labgic Technology Co., Ltd
CTAB	GEN-VIEW SCIENTIFIC INC	57-09-0(CAS)
Dropping	Jiangsu Songchang Medical Equipment Co., Ltd
Ethylenediaminetetraacetic acid, EDTA	Sinopharm Chemical Reagent Co.,Ltd	10009617
Forceps	LUXIANZI Biotechnology Co., Ltd
formaldehyde	Sinopharm Chemical ReagentCo., Ltd	10010018
Fully automatic sample fast grinder	Shanghai Jingxin Industrial Development Co., Ltd	Tissuelyser-96
glacial acetic acid	Sinopharm Chemical ReagentCo., Ltd	10000218
Grinding Tube	Shanghai Jingxin Industrial Development Co., Ltd
Isoamyl alcohol	Sinopharm Chemical Reagent Co.,Ltd	10003218
Isopropyl alcohol	Sinopharm Chemical Reagent Co.,Ltd	80109218
label	M&G Chenguang Stationery Co., Ltd.
Leica DMi8	Shanghai Leica Co.,Ltd	21903797
Magnesium sulfate heptahydrate, MgSO4	Sinopharm Chemical ReagentCo., Ltd	10013018
MICROSCOPE Cover glass	Zhejiang Shitai Industrial Co., Ltd
NaCl	Sinopharm Chemical Reagent Co.,Ltd	10019318
paper clips	M&G Chenguang Stationery Co., Ltd.
pencil	M&G Chenguang Stationery Co., Ltd.
pollinator brush	Shanghai Yimei Plastics Co., Ltd
Polyethylene glycol, PEG 6000	Beijing Dingguo Changsheng Biotechnology Co., Ltd	DH229-1
Polyethylene glycol, PEG-4000	Guangzhou saiguo biotech Co., Ltd	1521GR500
Potassium hydroxide, KOH	Sinopharm Chemical ReagentCo., Ltd	10017008
Potassium nitrate, KNO3	Sinopharm Chemical ReagentCo., Ltd	10017218
Scalpel	Jiangsu Songchang Medical Equipment Co., Ltd
Slide	Zhejiang Shitai Industrial Co., Ltd
Sodium hydroxide, NAOH	Sinopharm Chemical Reagent Co.,Ltd	10019718
Sucrose	Sinopharm Chemical ReagentCo., Ltd	10021418
sulfate paper	Taizhou Jinnong Mesh Factory
Thermostat water bath	Shanghai Jinghong Experimental Equipment Co., Ltd	L-909193
Trichloromethane	Sinopharm Chemical Reagent Co.,Ltd	10006818
Tripotassium phosphate tribasic trihydrate, K3PO4	Shanghai Lingfeng Chemical Reagent Co.,Ltd	20032318
Tris-HCl	GEN-VIEW SCIENTIFIC INC	1185-53-1
zip lock bags	M&G Chenguang Stationery Co., Ltd.
β-Mercaptoethanol	GEN-VIEW SCIENTIFIC INC	60-24-2(CAS)